CN105158469B - A kind of preparation method and application based on biotinylation amination Fe3O4 Yu the immunosensor of Streptavidin - Google Patents

Abstract

The invention belongs to the technical field of novel functional materials and biosensing detection, and provides a preparation method and application of an immunosensor based on biotinylated aminated Fe ₃ O ₄ and streptavidin. Specifically, an electrochemical immunosensor for detecting tumor marker antigens was prepared by using biotinylated Fe ₃ O ₄ as a label.

Description

An immunosensor based on biotinylated aminated Fe3O4 and streptavidin Preparation method and application

technical field

The invention belongs to the technical field of immune analysis and biosensing, and provides a preparation method and application of a biotinylated aminated Fe ₃ O ₄ and streptavidin multiple amplification immunosensor.

Background technique

The incidence of tumors is high and difficult to detect. The number of cases in my country is quite large, accounting for 55% of the number of cases in the world. Moreover, the growth and metastasis of tumors are fast, which poses great harm to human health. Sensitive detection of tumor markers, clinical early detection of tumors, screening of high-risk groups of tumors, differential diagnosis of benign and malignant tumors, judgment of tumor development, observation and evaluation of tumor treatment effects, and prediction of tumor recurrence and prognosis Prediction has a great impact and attracts a lot of people's attention.

At present, electrochemical immunosensors have been widely used in the detection of tumor markers. Sandwich-type electrochemical immunosensors combine high-specificity immunoassay technology and high-sensitivity electrochemical analysis technology, and have the advantages of high sensitivity, simple preparation, rapid detection and low cost. Low-level advantages have important application value in clinical testing, environmental monitoring, food safety control, biological monitoring and other fields.

The graphene used in the present invention is a wrinkled two-dimensional planar film, which has a large specific surface area, good electron transfer capability and catalytic performance, and can effectively adsorb and carry antibodies. The in-situ adsorption of SnO ₂ nanoparticles on the surface of graphene effectively avoids the stacking of graphene sheets, and the addition of polyaniline can greatly improve the electrochemical properties of SnO ₂ , and the presence of polyaniline can make SnO ₂ support graphite It is more strongly bonded to the antibody through the amino group.

Contents of the invention

The invention provides a preparation method and application of a biotinylated aminated Fe ₃ O ₄ and streptavidin multiple amplification immunosensor, which realizes ultrasensitive detection of tumor markers.

One of the objectives of the present invention is to provide a method for preparing an immunosensor of biotinylated aminated Fe ₃ O ₄ and streptavidin.

The second object of the present invention is to apply the prepared immunosensor of biotinylated aminated Fe ₃ O ₄ and streptavidin to highly sensitive and specific detection of tumor markers.

The technical solution of the present invention includes the following steps.

1. a kind of preparation method based on biotinylated aminated Fe ₃ O ₄ and the immunosensor of streptavidin, the steps are as follows:

(1) Polish the glassy carbon electrode with a diameter of 3 ~ 5 mm with Al ₂ O ₃ polishing powder, and clean it with ultrapure water;

(2) Take 6 µL, 0.5 ~ 1.5 mg/mL SnO ₂ loaded graphene polyaniline drop-coated on the surface of the electrode, dry at room temperature, rinse the surface of the electrode with ultrapure water, and dry in the air;

(3) Continue to add 6 µL, 8 ~ 12 µg/mL tumor marker capture antibody Ab ₁ dropwise to the surface of the electrode, rinse with ultrapure water, and dry in a refrigerator at 4°C;

(4) Continue to add 3 µL, 0.5 ~ 1.5 mg/mL bovine serum albumin BSA solution dropwise to the electrode surface, rinse the electrode surface with ultrapure water, and dry it in a refrigerator at 4 °C;

(5) Add 6 µL, 0.0005-10 ng/mL of a series of tumor marker antigen Ag solutions with different concentrations, rinse the surface of the electrode with ultrapure water, and dry it in a refrigerator at 4°C;

(6) Apply 6 µL, 1-3 mg/mL biotinylated tumor marker detection antibody B-Ab ₂ solution dropwise on the surface of the electrode, and place it in a refrigerator at 4°C to dry;

(7) Apply 6 µL, 0.1-0.3 mg/mL streptavidin solution onto the surface of the electrode, and place it in a refrigerator at 4°C to dry;

(8) 6 µL, 1 ~ 3 mg/mL biotinylated aminated Fe ₃ O ₄ solution, drop-coated on the surface of the electrode, placed in a 4 ° C refrigerator to dry, prepared a biotinylated amino-based Immunosensor of Fe ₃ O ₄ and streptavidin.

2. Preparation of SnO ₂ loaded graphene polyaniline, biotinylated aminated Fe ₃ O ₄ , and biotinylated tumor marker detection antibody B-Ab ₂ solution

(1) Preparation of _SnO2 supported graphene polyaniline

Weigh 40-60 mg of _SnO2 -loaded graphene, measure 10 mL of N,N-dimethylformamide, mix them in a 50-mL beaker, continue to add 0.04-0.06 mL of aniline, 1-2 mL, the mass fraction is 37% hydrochloric acid HCl solution, magnetically stirred for 1 h, then weighed 0.05 ~ 0.15 g of ammonium persulfate into the above beaker, and ultrasonically reacted at room temperature for 12 h; centrifuged to obtain SnO ₂ loaded graphene polyaniline;

(2) Preparation of biotinylated aminated Fe ₃ O ₄

① Synthesis of aminated Fe ₃ O ₄

Weigh 0.5 ~ 1.5 g Fe ₃ O ₄ and dissolve it in 50 ml of absolute ethanol, ultrasonicate for 1 h, add 2 ~ 6 g of 3-aminopropyltriethoxysilane at a constant temperature of 40°C under stirring conditions, stir Time 8 h; after magnetic separation, washed three times with water, washed three times with ethanol, and dried at room temperature to obtain aminated Fe ₃ O ₄ ;

②Synthesis of biotinylated aminated Fe ₃ O ₄

Dissolve 1.5–2.5 mg of biotin-aminoacetic acid n-hydroxysuccinimide in 1 ml of N,N _- dimethylformamide, then add 10–30 mg of aminated magnetic _Fe3O4 and 30 mL of pH7. 4 in phosphate buffer solution, stirred at 37°C for 12 h, washed three times with N,N-dimethylformamide and water respectively, and dried at room temperature to obtain biotinylated aminated Fe ₃ O ₄ ;

(3) Preparation of biotinylated tumor marker detection antibody B-Ab ₂ solution

Dissolve 1-3 mg of biotin-glycolic acid n-hydroxysuccinimide in 1 mL of pH 7.4 phosphate buffer solution, shake to dissolve, then add 100 µL, 80-120 µg/mL tumor marker detection antibody Solution and 900 µL, 50 mmol/L phosphate buffer solution of pH 7.4, oscillating in a constant temperature shaking incubator at 4°C, and incubating for 12 h to prepare a biotinylated tumor marker detection antibody B-Ab ₂ solution, at 4°C Save it for future use.

3. Detection of tumor markers

(1) The electrochemical workstation was used to test with a three-electrode system. The saturated calomel electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared sensor was used as the working electrode. 8.0 for testing in phosphate buffered saline solution;

(2) The time-current method was used to detect the analyte, the input voltage was -0.4 V, the sampling interval was 0.1 s, and the running time was 400 s;

(3) When the background current tended to be stable, 10 µL, 5 mol/L hydrogen peroxide solution was injected into 10 mL, 50 mmol/L pH 7.4 phosphate buffer solution every 50 s, and the current change was recorded.

The tumor markers mentioned above are selected from one of the following: AFP, CEA, PSA

The raw materials used in the present invention can be purchased in chemical reagent companies or biopharmaceutical companies.

Beneficial results of the present invention

(1) The present invention uses _SnO2 loaded graphene polyaniline. Graphene has a large specific surface area, which can increase the binding sites of antibodies. Graphene is oxidized to reduce graphene, which is a hydrophilic substance and has excellent Dispersion, and the carboxyl group can be effectively combined with the amino group on the antibody. The in-situ reduction of SnO ₂ nanoparticles on the graphene sheet can effectively avoid the stacking of graphene sheets. The addition of polyaniline can greatly improve the electrochemical performance of SnO ₂ properties, and the presence of polyaniline can increase the antibody binding rate, making the binding more firm;

(2) Using biotinylated aminated Fe ₃ O ₄ as the capture antibody label, Fe ₃ O ₄ nanoparticles have extremely high strength and good conductivity, and have a catalytic effect on hydrogen peroxide, and Fe ₃ O ₄ After amination, it can effectively bind biotin. The cross-type streptavidin has four binding sites, which can be combined with four biotins at the same time, one of which is bound to a biotinylated secondary antibody, and the other three are bound to a biotinylated capture antibody label substances, realizing the triple amplification of electrochemical signals, thereby improving the sensitivity of the sensor and reducing the detection limit;

(3) An immunosensor based on biotinylated aminated Fe ₃ O ₄ and streptavidin can detect tumor markers with a linear range of 0.0005 ~ 10 ng/mL and a detection limit of 0.1 pg/mL, indicating that An immunosensor based on biotinylated aminated Fe ₃ O ₄ and streptavidin can achieve the purpose of accurate determination.

detailed description

Example 1 Preparation of an immunosensor based on biotinylated aminated Fe ₃ O ₄ and streptavidin

(1) Polish the glassy carbon electrode with a diameter of 3 mm with Al ₂ O ₃ polishing powder, and clean it with ultrapure water;

(2) Take 6 µL, 0.5 mg/mL SnO ₂ loaded graphene polyaniline drop-coated on the electrode surface, dry it at room temperature, rinse the electrode surface with ultrapure water, and dry it;

(3) Continue to drop 6 µL, 8 µg/mL tumor marker capture antibody Ab ₁ onto the surface of the electrode, rinse with ultrapure water, and dry in a refrigerator at 4°C;

(4) Continue to add 3 µL, 0.5 mg/mL bovine serum albumin BSA solution dropwise to the electrode surface, rinse the electrode surface with ultrapure water, and dry it in a refrigerator at 4 °C;

(5) Add 6 µL, 0.0005-10 ng/mL of a series of tumor marker antigen Ag solutions with different concentrations, rinse the surface of the electrode with ultrapure water, and dry it in a refrigerator at 4°C;

(6) Apply 6 µL, 1 mg/mL biotinylated tumor marker detection antibody B-Ab ₂ solution dropwise on the surface of the electrode, and place it in a refrigerator at 4°C to dry;

(7) Apply 6 µL, 0.1 mg/mL streptavidin solution dropwise on the surface of the electrode, and place it in a refrigerator at 4°C to dry;

(8) Apply 6 µL, 1 mg/mL biotinylated aminated Fe ₃ O ₄ solution onto the surface of the electrode, and place it in a refrigerator at 4°C to dry to prepare a biotinylated aminated Fe 3 O 4 solution. Immunosensor of ₃ O ₄ and streptavidin.

Example 2 Preparation of an immunosensor based on biotinylated aminated Fe ₃ O ₄ and streptavidin

(1) Polish the glassy carbon electrode with a diameter of 4 mm with Al ₂ O ₃ polishing powder, and clean it with ultrapure water;

(2) Take 6 µL, 1.0 mg/mL SnO ₂ loaded graphene polyaniline drop-coated on the electrode surface, dry at room temperature, rinse the electrode surface with ultrapure water, and dry;

(3) Continue to add 6 µL, 10 µg/mL tumor marker capture antibody Ab ₁ dropwise to the surface of the electrode, rinse with ultrapure water, and dry in a refrigerator at 4°C;

(4) Continue to add 3 µL, 1.0 mg/mL bovine serum albumin BSA solution dropwise to the electrode surface, rinse the electrode surface with ultrapure water, and dry it in a refrigerator at 4 °C;

(5) Add 6 µL, 0.0005-10 ng/mL of a series of tumor marker antigen Ag solutions with different concentrations, rinse the surface of the electrode with ultrapure water, and dry it in a refrigerator at 4°C;

(6) Apply 6 µL, 2 mg/mL biotinylated tumor marker detection antibody B-Ab ₂ solution dropwise on the surface of the electrode, and place it in a refrigerator at 4°C to dry;

(7) Apply 6 µL, 0.2 mg/mL streptavidin solution dropwise on the surface of the electrode, and place it in a refrigerator at 4°C to dry;

(8) Apply 6 µL, 2 mg/mL biotinylated aminated Fe ₃ O ₄ solution onto the surface of the electrode, and place it in a refrigerator at 4°C to dry to prepare a biotinylated aminated Fe 3 O 4 solution based on biotinylated aminated Fe 3 O 4 Immunosensor of ₃ O ₄ and streptavidin.

Example 3 Preparation of an immunosensor based on biotinylated aminated Fe ₃ O ₄ and streptavidin

(1) Polish the glassy carbon electrode with a diameter of 5 mm with Al ₂ O ₃ polishing powder, and clean it with ultrapure water;

(2) Take 6 µL, 1.5 mg/mL SnO ₂ loaded graphene polyaniline drop-coated on the electrode surface, dry at room temperature, rinse the electrode surface with ultrapure water, and dry;

(3) Continue to add 6 µL, 12 µg/mL tumor marker capture antibody Ab ₁ dropwise to the surface of the electrode, rinse with ultrapure water, and dry in a refrigerator at 4°C;

(4) Continue to add 3 µL, 1.5 mg/mL bovine serum albumin BSA solution dropwise to the electrode surface, rinse the electrode surface with ultrapure water, and dry it in a refrigerator at 4 °C;

(5) Add 6 µL, 0.0005-10 ng/mL of a series of tumor marker antigen Ag solutions with different concentrations, rinse the surface of the electrode with ultrapure water, and dry it in a refrigerator at 4°C;

(6) Apply 6 µL, 3 mg/mL biotinylated tumor marker detection antibody B-Ab ₂ solution dropwise on the surface of the electrode, and place it in a refrigerator at 4°C to dry;

(7) Apply 6 µL, 0.3 mg/mL streptavidin solution dropwise on the surface of the electrode, and place it in a refrigerator at 4°C to dry;

(8) Apply 6 µL, 3 mg/mL biotinylated aminated Fe ₃ O ₄ solution dropwise on the surface of the electrode, and place it in a refrigerator at 4°C to dry to prepare a biotinylated aminated Fe 3 O 4 solution based on biotinylated aminated Fe 3 O 4 Immunosensor of ₃ O ₄ and streptavidin.

Example 4 Preparation of SnO ₂ loaded graphene polyaniline, biotinylated aminated Fe ₃ O ₄ , biotinylated tumor marker detection antibody B-Ab ₂ solution

(1) Preparation of _SnO2 supported graphene polyaniline

Weigh 40 mg of _SnO supported graphene, measure 10 mL of N,N-dimethylformamide, mix in a 50 mL beaker, continue to add 0.04 mL of aniline, 1 mL of 37% hydrochloric acid HCl solution, Magnetically stirred for 1 h, then weighed 0.05 g of ammonium persulfate and added to the above-mentioned beaker, and ultrasonically reacted for 12 h at room temperature; centrifuged to obtain _SnO Loaded graphene polyaniline;

(2) Preparation of biotinylated aminated Fe ₃ O ₄

① Synthesis of aminated Fe ₃ O ₄

Weigh 0.5 g Fe ₃ O ₄ and dissolve it in 50 ml of absolute ethanol, ultrasonicate for 1 h, add 2 g of 3-aminopropyltriethoxysilane at a constant temperature of 40°C under stirring, and stir for 8 h; After magnetic separation, washing three times with water, three times with ethanol, and drying at room temperature, aminated Fe ₃ O ₄ was obtained;

②Synthesis of biotinylated aminated Fe ₃ O ₄

Dissolve 1.5 mg of biotinylated glycine n-hydroxysuccinimide in 1 ml of N,N-dimethylformamide, then add ₁₀ mg of aminated magnetic _Fe3O4 and 30 mL of phosphate at pH7.4 buffer solution, stirred at 37°C for 12 h, washed three times with N,N-dimethylformamide and water respectively, and dried at room temperature to obtain biotinylated aminated Fe ₃ O ₄ ;

(3) Preparation of biotinylated tumor marker detection antibody B-Ab ₂ solution

Dissolve 1 mg of biotin-aminoacetic acid n-hydroxysuccinimide in 1 mL of pH 7.4 phosphate buffer solution, shake to dissolve, then add 100 µL, 80 µg/mL tumor marker detection antibody solution and 900 µL , 50 mmol/L phosphate buffer solution of pH 7.4, oscillate in a constant temperature shaking incubator at 4°C, and incubate for 12 h to prepare a biotinylated tumor marker detection antibody B-Ab ₂ solution, and store it at 4°C for future use .

Example 5 Preparation of SnO ₂ loaded graphene polyaniline, biotinylated aminated Fe ₃ O ₄ , biotinylated tumor marker detection antibody B-Ab ₂ solution

(1) Preparation of _SnO2 supported graphene polyaniline

Weigh 50 mg of _SnO2 -loaded graphene, measure 10 mL of N,N-dimethylformamide, mix in a 50 mL beaker, continue to add 0.05 mL of aniline, 1 ~ 2 mL of hydrochloric acid with a mass fraction of 37% HCl solution, magnetically stirred for 1 h, then weighed 0.10 g ammonium persulfate and added to the above beaker, and ultrasonically reacted at room temperature for 12 h; centrifuged to obtain SnO ₂ loaded graphene polyaniline;

(2) Preparation of biotinylated aminated Fe ₃ O ₄

① Synthesis of aminated Fe ₃ O ₄

Weigh 1.0 g Fe ₃ O ₄ and dissolve it in 50 ml of absolute ethanol, ultrasonicate for 1 h, add 4 g of 3-aminopropyltriethoxysilane at a constant temperature of 40°C under stirring, and stir for 8 h; After magnetic separation, washing three times with water, three times with ethanol, and drying at room temperature, aminated Fe ₃ O ₄ was obtained;

②Synthesis of biotinylated aminated Fe ₃ O ₄

Dissolve 2.0 mg of biotinylated glycine n-hydroxysuccinimide in 1 ml of N,N-dimethylformamide, then add ₂₀ mg of aminated magnetic _Fe3O4 and 30 mL of phosphate at pH7.4 buffer solution, stirred at 37°C for 12 h, washed three times with N,N-dimethylformamide and water respectively, and dried at room temperature to obtain biotinylated aminated Fe ₃ O ₄ ;

(3) Preparation of biotinylated tumor marker detection antibody B-Ab ₂ solution

Dissolve 2 mg of biotin-aminoacetic acid n-hydroxysuccinimide in 1 mL of pH 7.4 phosphate buffer solution, shake to dissolve, then add 100 µL, 100 µg/mL tumor marker detection antibody solution and 900 µL , 50 mmol/L phosphate buffer solution of pH 7.4, oscillate in a constant temperature shaking incubator at 4°C, and incubate for 12 h to prepare a biotinylated tumor marker detection antibody B-Ab ₂ solution, and store it at 4°C for future use .

Example 6 Preparation of SnO ₂ loaded graphene polyaniline, biotinylated aminated Fe ₃ O ₄ , biotinylated tumor marker detection antibody B-Ab ₂ solution

(1) Preparation of _SnO2 supported graphene polyaniline

Weigh 60 mg of _SnO2 -loaded graphene, measure 10 mL of N,N-dimethylformamide, mix in a 50 mL beaker, continue to add 0.06 mL of aniline, 2 mL of 37% hydrochloric acid HCl solution , magnetically stirred for 1 h, then weighed 0.15 g of ammonium persulfate and added to the above-mentioned beaker, and ultrasonically reacted for 12 h at room temperature; centrifuged to obtain SnO ₂ loaded graphene polyaniline;

(2) Preparation of biotinylated aminated Fe ₃ O ₄

① Synthesis of aminated Fe ₃ O ₄

Weigh 1.5 g Fe ₃ O ₄ and dissolve it in 50 ml of absolute ethanol, ultrasonicate for 1 h, add 6 g of 3-aminopropyltriethoxysilane at a constant temperature of 40°C under stirring, and stir for 8 h; After magnetic separation, washing three times with water, three times with ethanol, and drying at room temperature, aminated Fe ₃ O ₄ was obtained;

②Synthesis of biotinylated aminated Fe ₃ O ₄

Dissolve 2.5 mg of biotinylated glycine n-hydroxysuccinimide in 1 ml of N,N _- dimethylformamide, then add 30 mg of aminated magnetic _Fe3O4 and 30 mL of phosphate at pH7.4 buffer solution, stirred at 37°C for 12 h, washed three times with N,N-dimethylformamide and water respectively, and dried at room temperature to obtain biotinylated aminated Fe ₃ O ₄ ;

(3) Preparation of biotinylated tumor marker detection antibody B-Ab ₂ solution

Dissolve 3 mg of biotin-aminoacetic acid n-hydroxysuccinimide in 1 mL of pH 7.4 phosphate buffer solution, shake to dissolve, then add 100 µL, 120 µg/mL tumor marker detection antibody solution and 900 µL , 50 mmol/L phosphate buffer solution of pH 7.4, oscillate in a constant temperature shaking incubator at 4°C, and incubate for 12 h to prepare a biotinylated tumor marker detection antibody B-Ab ₂ solution, and store it at 4°C for future use .

Example 7 Detection of Tumor Marker AFP

(1) The electrochemical workstation was used to test with a three-electrode system. The saturated calomel electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared sensor was used as the working electrode. 8.0 for testing in phosphate buffered saline solution;

(2) The time-current method was used to detect the analyte, the input voltage was -0.4 V, the sampling interval was 0.1 s, and the running time was 400 s;

(3) When the background current tends to be stable, inject 10 µL, 5 mol/L hydrogen peroxide solution into 10 mL, 50 mmol/L pH7.4 phosphate buffer solution every 50 s, and record the current change;

(4) According to the linear relationship between the obtained current intensity and AFP concentration, the working curve was drawn, and the measured linear range was 0.0005 ~ 10 ng/mL, and the detection limit was 0.1 pg/mL.

The detection of embodiment 8 tumor marker CEA

According to the method of Example 7, CEA in the sample was detected, the linear range was 0.001-10 ng/mL, and the detection limit was 0.2 pg/mL.

The detection of embodiment 9 tumor marker PSA

According to the method of Example 7, the PSA in the sample was detected, the linear range was 0.0005-10 ng/mL, and the detection limit was 0.1 pg/mL.

Claims (3)

1. one kind based on biotinylation amination Fe₃O₄With the preparation method of the immunosensor of Streptavidin, its feature exists In, step is as follows:

(1) by the glass-carbon electrode Al of a diameter of 3 ~ 5 mm₂O₃Polishing powder is polished, and ultra-pure water cleans up；

(2) 6 L, the SnO of 0.5 ~ 1.5 mg/mL are taken₂Load Graphene polyaniline drop coating is dried under electrode surface, room temperature, uses Ultrapure water electrode surface, dries；

(3) continue the tumor markers of 6 L, 8 ~ 12 g/mL is captured antibody A b₁Being added drop-wise to electrode surface, ultra-pure water rushes Wash, 4 DEG C of refrigerators are dried；

(4) continuing to be added drop-wise to the bovine serum albumin BSA solution of 3 L, 0.5 ~ 1.5 mg/mL electrode surface, ultra-pure water rushes Wash electrode surface, 4 DEG C of refrigerators dry；

(5) the tumor markers antigen A g solution of a series of variable concentrations of 6 L, 0.0005 ~ 10 ng/mL is dripped, ultrapure Water rinses electrode surface, is dried in 4 DEG C of refrigerators；

(6) by 6 L, the biotinylated tumor-marker analyte detection antibody B-Ab of 1 ~ 3 mg/mL₂Solution, drop coating is in electrode table On face, it is placed in 4 DEG C of refrigerators and dries；

(7) by 6 L, the solution of streptavidin of 0.1 ~ 0.3 mg/mL, drop coating, on electrode surface, is placed in 4 DEG C of refrigerators Dry；

(8) by 6 L, biotinylation amination Fe of 1 ~ 3 mg/mL₃O₄Solution, drop coating, on electrode surface, is placed in 4 DEG C of ice Case dries, prepares a kind of based on biotinylation amination Fe₃O₄Immunosensor with Streptavidin.

2. as claimed in claim 1 a kind of based on biotinylation amination Fe₃O₄System with the immunosensor of Streptavidin Preparation Method, described SnO₂Load Graphene polyaniline, biotinylation amination Fe₃O₄, biotinylated tumor-marker analyte detection resist Body B-Ab₂The preparation of solution, step is as follows:

(1) SnO₂The preparation of load Graphene polyaniline

Weigh the SnO of 40 ~ 60 mg₂Load Graphene, measures 10 mL DMFs, is mixed in 50 mL beakers In, continuously adding 0.04 ~ 0.06 mL aniline, 1 ~ 2 mL, mass fraction is the hydrochloric acid HCl solution of 37%, magnetic agitation 1 H, after weigh 0.05 ~ 0.15 g Ammonium persulfate. and add in above-mentioned beaker, ultrasonic reaction 12 h under room temperature；It is centrifugally separating to obtain SnO₂Load Graphene polyaniline；

(2) biotinylation amination Fe₃O₄Preparation

1. amination Fe₃O₄Synthesis

Weigh 0.5 ~ 1.5 g Fe₃O₄It is dissolved in 50 ml dehydrated alcohol, ultrasonic time 1 h, at constant temperature 40 DEG C, stirring condition The 3-aminopropyl triethoxysilane of lower addition 2 ~ 6 g, mixing time 8 h；Rear Magneto separate, washes three times, washing with alcohol three Secondary, drying at room temperature, prepare amination Fe₃O₄；

2. biotinylation amination Fe₃O₄Synthesis

1.5 ~ 2.5 mg biotin glycine n-N-Hydroxysuccinimide are dissolved in the DMF of 1 ml, It is subsequently adding 10 ~ 30 mg amination magnetic Fe₃O₄With the phosphate buffered solution of 30 mL pH7.4, at 37 DEG C, stir 12 h, Carry out three washings, drying at room temperature with DMF and water respectively, prepare biotinylation amination Fe₃O₄；

(3) biotinylated tumor-marker analyte detection antibody B-Ab₂The preparation of solution

1 ~ 3 mg biotin glycine n-N-Hydroxysuccinimide is dissolved in the phosphate buffered solution of the pH 7.4 of 1 mL In, concussion dissolve, add 100 μ L, the tumor-marker analyte detection antibody-solutions of 80 ~ 120 μ g/mL and 900 μ L, 50 The phosphate buffered solution of the pH7.4 of mmol/L, vibrates in 4 DEG C of constant-temperature shaking incubators, hatches 12 h, prepares biotinylated Tumor-marker analyte detection antibody B-Ab₂Solution, saves backup at 4 DEG C.

3. the one that prepared by preparation method as claimed in claim 1 is based on biotinylation amination Fe₃O₄With Streptavidin Immunosensor, for the detection of tumor markers, detecting step is as follows:

(1) using electrochemical workstation to test with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is Auxiliary electrode, prepared sensor is working electrode, molten in pH 5.0 ~ 8.0 phosphate-buffered of 10 mL, 50 mmol/L Liquid is tested；

(2) analyte is detected by used time m-current method, and input voltage is-0.4 V, sampling interval 0.1 s, runs the time 400 s；

(3) after background current tends towards stability, every 50 s to 10 mL, the phosphate buffered solution of the pH7.4 of 50 mmol/L Middle injection 10 μ L, the hydrogen peroxide solution of 5 mol/L, record current changes.